Pyridine dinucleotide transhydrogenase of the inner mitochondrial membrane catalyzes a reaction between oxidized and reduced forms of NAD and NADP that is coupled to proton translocation and membrane energization. Thus, the following reaction represents a fourth classical energy-coupling site of the respiratory chain: nH(plus)(in) plus NADPH plus NAD(plus) yield nH(plus)(out) plus NADP(plus) plus NADH. It is proposed to investigate the mechanism of energy coupling by (a) incorporation of previously purified bovine heart transhydrogenase into synthetic proteoliposomes and analyzing for transhydrogenase-dependent proton-pumping, (b) localizing the enzyme in the native membrane by chemical modification, cross-linking and immunological techniques, (c) characterization of the molecular weight, subunit structure and putative reduced intermediate of pure enzyme, (d) specific chemical modification of membrane-bound and pure enzyme to elucidate catalytic amino acid residues, and those potentially in a proton binding domain, (e) determining the stoichiometry of proton translocation coupled to transhydrogenation, (f) further purification and characterization of the Rhodospirillum rubrum transhydrogenase system. Studies on the regulation of mitochrondrial one-carbon metabolism by transhydrogenation are also proposed.